MicroRNA (miRNA) expression profiling in the retina, retinal endothelial cells (RECs) and vitreous humor of streptozotocin (STZ)-induced diabetic rats are described herein for the first time and compared to profiling in non-diabetic rats. A number of miRNAs that are involved in multiple pathological pathways of diabetic retinopathy (DR) were identified in the retina and REC for use as therapeutic targets for the treatment of diabetic retinopathy (DR), and in the vitreous humor for use as biomarkers for diagnosis of DR and personalized medicine.
DR is the leading cause of blindness in the industrialized world in people between the ages of 25 and 74. Nearly all individuals who have had type I diabetes for more than 15 years develop DR. Approximately 50-80% of type II diabetic patients also develop retinopathy after 20 years of diabetes. DR is a result of interplays of multiple pathogenetic processes caused by hyperglycemia and abnormalities of insulin signaling pathways, including retinal microvascular dysfunction, abnormal inflammatory responses, and neuroretinal dysfunction and degeneration. In diabetic patients, hyperglycemia induces increased production of reactive oxygen species (ROS), formation of advanced glycation end-product (AGE), flux through polyol and hexamine pathways and activation of protein kinase C, and results in early reduction of retinal blood flow, leukostasis, vaso-occlusion, proinflammatory responses, endothelial-cell (EC) death, pericyte and vascular smooth muscle drop-out and microaneurysms, leading to a breakdown of the blood-retina barrier (BRB) and increased permeability. Early damage to retinal microvasculature plays pivotal roles in the development of DR. Leakage of fluid into the central retina results in diabetic macular edema and ischemic hypoxia, which promotes neovascularization in the attempt to restore blood flow. The newly-formed vessels may destroy normal retinal architecture and cause bleeding in the eyes and, ultimately, impair vision. Laser coagulation treatment is still the mainstay in management of DR, however, it may break down the BRB and worsen macular edema. Although significant progress has been made involving blocking the activity of vascular endothelial growth factor (VEGF) and pancreatic islet transplantation, the long-term effects of these treatments need to be evaluated, and other approaches to the treatment still are to be explored.
Although originally identified as a regulator of κB light chain expression in mature B and plasma cells, NF-κB has been shown to be expressed in almost all cell types, mediating responses to a remarkably diverse external and internal stimuli. NF-κB is a pivotal element of wide-range basic cellular functions, including proliferation, differentiation, survival and migration; and physiological and pathological processes, including inflammation, immunity, angiogenesis, stress response, neurogenesis, neural plasticity, learning and memory. Dysregulation of NF-κB pathways plays important roles in inflammatory and immune defects and related diseases, including autoimmunity, diabetes and its microvascular complications, atherosclerosis, as well as neurodegenerative diseases, and cancer development.
Numerous pathways lead to the activation of NF-κB, including G-protein couple receptor (GPCR)-mediated NF-κB activation. GPCRs have a characteristic seven-transmembrane-domain structure, and constitute one of the largest families of cell surface receptor proteins. GPCRs are expressed in all organ systems throughout the body, and transduce diverse extracellular signals, including hormones, neurotransmitters, light, odorants, tastants, chemokines and calcium, playing pivotal roles in wide-range biological functions. Many GPCR ligands, including thrombin, lysophosphatidic acid (LPA), angiotensin II, endothelin-1 (ET-1), platelet activating factor (PAF), IL-8 (CXCL-8) and stromal cell-derived factor (SDF; or CXCL12), etc, induces NF-κB activation, promoting inflammatory reactions and other cellular functions in many cell types and organ systems. GPCR engagement with ligands initiates intracellular recruitment of heterotrimeric guanine nucleotide-binding proteins (G-proteins) to the receptors. G proteins consist of three subunits, the Gα subunits, which responsible for GTP/GDP binding and GTP hydrolysis, and β and γ subunits (Gβ/γ). Activated G proteins induce PKC-involved signal transduction pathways, which converge to IKK and NF-κB activation. Caspase-recruitment domain (CARD)-containing scaffold/adaptor proteins, Caspase Recruitment Domain family member 10 (CARD10) and B-cell lymphoma 10 (Bc110), and caspase-like protein mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) have been shown to form CARD10-Bc110-MALT′ complex and mediate GPCR-induced NF-κB activation between PKC and IKK and NF-κB activation.
microRNAs (miRNAs) are small, non-coding, regulatory RNAs, and represent a newly-recognized level of gene-expression regulation. It is estimated that there are more than 800-1000 miRNAs in the human genome and that more than one-third of the protein-coding genes in the human genome are subjected to miRNA regulation. Defects in miRNA biogenesis and mutations in miRNAs and the target sites of their downstream target mRNAs may cause diseases in animals and humans. miRNAs have been shown to be involved in many aspects of NF-κB activation pathways. miR-146 is shown to be induced by a variety of microbial components and proinflammatory cytokines in NF-κB-dependent manner, and in turn, miR-146 inhibits interleukin 1 receptor (IL-1R)/Toll-like receptor (TLR)-mediated NF-κB activation pathway by targeting two key adaptor molecules of this pathway, IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6), in monocytes, suggesting a novel mechanism of negative feedback regulation on IL-1R/TLR-mediated NF-κB activation. The roles of miRNAs in DR, DR patients and diabetic animal models are not established.
In spite of these reports, roles of miRNAs in GPCR-mediated NF-κB activation are still unknown.